Figure 4. Cell-type-specific multicolor STORM imaging illuminates homogeneous nanoscale distribution of CB₁ receptors on GABAergic axon terminals.

(a,d) Deconvolved confocal images show a biocytin-filled CA1 pyramidal neuron and adjacent CB₁-positive boutons. (b,e) Putative afferents of the labeled neuron are identified. (c,f) Dual-channel 3D-STORM demonstrates accumulation of the active zone protein bassoon at the contact site, indicative of a perisomatic and dendritic afferent synapses. Representative images are shown from experiments repeated on 10 biocytin-filled pyramidal neurons. (g,i) Confocal images of axon terminals of biocytin-filled perisomatic and dendritic interneurons, respectively. (h,j) Dual-channel 3D-STORM reveals the nanoscale bassoon and CB₁ distribution. (k,m) Localization points are visualized in 3D. The Euclidean distances between CB₁ and bassoon LPs can be readily calculated. (l,n) To allow intermolecular distance measurements along the structure surface, the shortest paths between CB₁ and bassoon LPs on the bouton surface were determined after fitting a 3D-convex hull on the CB₁ localization points. (o) Neither the Euclidean nor the surface distance is different between axon terminals of perisomatic and dendritic interneurons (n = 311 and 141 boutons, Mann-Whitney U test, p = 0.69, U = 21420 for Euclidean distance; n = 33 and 22 boutons, p = 0.31, U = 305 for surface distance). Graphs show raw data and median±IQR. (p) The density of receptors (CB₁ NLP within 200 nm from random points of the surface, median±IQR) is plotted against surface distance from bassoon. Perisynaptic (within 250 nm) and extrasynaptic (1000-1250 nm) CB₁ densities are similar within boutons, and between cell types.